TOXICOLOGY AND APPLIED PEIARMACOLOOY 7, 93-101 (1965)



### Comparative Eye Testing: Man vs. Animal

JOHN HENRY BECKLEY
Medical Research Services, Colgate-Palmolice Company, Sew York, New York lOOZ-7
The title of this paper LLComparative Eye Testing: Man VUS. Animal” should be modified to read, ‘Some Observations and Limited Conclusions Which Might Be Acceptable, after Review of Data Obtained by Following the Recommended Procedure for Tests for Irritancy of Substances to the Eye Mucosa of Albino Rabbits, and
aComparison of the Results with the SameSubstances in the Eyes of Dogs, Monkeys, and Man.” I shudder to think what the Program Committee’s reaction would have beenhad I sent them this title! Because of our concern with making products safe under recommended conditions of use and reasonably foreseeable misuse, we are vitally interested in the development of the most meaningful and practical tests of safety, both in vitro and in ZJ~VO, that canbedevised. We must keep in mind that aproduct’s physical form, container. and labeling also enter into this problem of safety. Any discussion of the toxicology of the eye must include reference to W. Morton Grant. His book Toxicology of the Eye (1962) is all-inclusive. It is not only one of the most informative works in this field, but it is delightful reading. The preface states: (‘The purpose of this book is topresent asynopsis of what we know about substances which have toxic properties injurious to the eyes, disturbing to vision or affecting the eyes in other unwanted ways.” The information which is summarized represents what has been published in approximately the last hundred years, plus a moderate number of previously unpublished observations. Approximately 1600 substances are covered, with previously unpublished observations on more than 200. The preface continues: “Intentionally in this book the weaknesses and deficiencies of our knowledge are made quite evident, so that anyone having an opportunity to make further observations may easily see where the need is for more complete and accurate information. “In the same vein, in the interest of research and investigation, attention is called to unsolved problems of mechanism of injurious action and to conditions for which specific treatments are lacking, yet by scientific methods could conceivably be developed.”
Mechanism of Injurious Action When we review the literature of the past several years, it becomes apparent that
our knowledge of the mechanism of “injurious action” on the eye from direct contact with toxic substances is wanting. The annual review of toxicolo,T and pharmacology which appears in the Archives of Ophthalmology has not in the past three years made any mention of toxic substances accidentally introduced into the eye. (Who is better qualified than the ophthalmologist to observe and help explain the mechanism of injury caused by substances which enter the eye accidentally or otherwise?) Much

9394 JOHN H. BECKLEY

work has beendonewith various species of animals to test products for potential injury to the eyes, but little has beenwritten on the relation of such findings to human eyes. Are the results obtained in animal eyes truly indicative of what will happen in human eyes? Are there significant differences in the anatomy, histology, and physiology of rabbit, dog, monkey, and human eyes? The anatomist will say “yes,” and sowill the histologist and physiologist. But are these differences of a kind and magnitude to invalidate results obtained with these animals as predictive of effects in human eyes? How dothe rabbit, dog, and monkey eyes differ from each other and from the human eye?Not asawhole, but in terms of the particular tissues that may bedirectly involved following the introduction of irritant substances into the eye. This will narrow our discussion principally to a consideration of the lids, conjunctiva, cornea, and lacrimal apparatus and accessory glands. There are some interesting facts relating to the eyes of these animals which the comparative anatomist has assembled, and which to a few of us may not be known. These three animals, for the purpose of classification, are referred to by the zoologists
as “placentals.” They are all mammals. The rabbit is one of the most widely used test animals. It is classified among the Lagomorpha, an order of gnawing mammals resembling the Rodentia, but having two pair of upper incisors, onebehind the other. The dog belongs to alarge amorphous order, the Carnivora (flesh eaters) and the monkey to the order of Primates. And so the three test animals about which we are talking are all ‘Lplacentals.” They are all mammals, but each belongs to a different order of mammals. Throughout the many orders of placentals, a considerable range of variation in the structure of the eye and adnexa occurs, but through the class the similarity is great. For instance, the histological structure of the cornea is built on the typical vertebrate plan as seen in man, but most species do not have a Bowman’s membrane. Descemet’s membrane, however, ispresent including its endothelium, which is often very substantial (Prince et at., 1961). The thickness and surface area of the cornea1 epithelium vary considerably. Most placentals have lacrimal glands, situated in the upper outer quadrant, which secrete a watery fluid. However, in other types of animals the lacrimal gland is not always associated with the upper lid; for instance, in the t.errestrial amphibian it is near the medial canthus associated with the lower lid. In birds and reptiles it is near the outer canthus, but still associated with the lower lid. In the mouse family the gland is said to belacking. In the pig its secretion ismucoid rather than watery, and inthe whale andporpoise it isoily. In addition to the lacrimal glands, there are two other glands which are commonly found in placentals: (1) Harder’s gland situated on the nasal side of the orbit whose function is to lubricate the nictitating membrane, and (2) the nictitan gland situated around the cartilage within the nictitating membrane. We were unable to find comparative data reIating to the tissues in which we are most interested for the purpose of this discussion, namely, the similarity or dissimilarity of the conjunctiva, the cornea, and the lacrimal and accessory glands in the rabbit, the dog, and the monkey. All three of these animals have awell-developed palpebral and bulbar conjunctiva. The arrangement isvery similar to that found in the human. There are abundant blood and lymph capillaries, and this explains the rapidity with which certain substances are absorbed from the conjunctival sac. ICOMPARATIVE EYE TESTING 9.5

have been told that there are compounds, one drop of which when introduced into the conjunctival sac of the rabbit, will cause almost instantaneous death. I understand that some organic phosphates will act in this manner when dropped into the eye of a rabbit. The mystery story writers should have a field-day with this if they ,learn about it. It will surely top Shakespeare’s account of the sudden death of Ham let’s father by henbane which was dropped into his ear while he was sleeping in the orchard. But then this was only a“ghost’s story.”

Lacrimal and Accessory Glands of the Rabbit, Dog, and Monkey How frequently many of ushave heardthat the reasonwhy the rabbit isnot a goodtestanimalfor determiningeyeirritancy isthat the rabbit doesnot tear. This statement,of course,mustbequalified. The rabbit hasawell-definedlacrimal gland almostcompletelysurroundingtheorbital rim. There isaductwhichpasses into the conjunctiva of the upperlid near the lateral canthus. It is reported that there is alsoavery smallductwhich passes into the lowerlid. This gland isa serousgland with little vascularization. In addition to the lacrimal gland, the rabbit alsohasa well-developedHarder’s glandwhich secreteslipids, and anictitan glandwhich hasseroussecretingunits. Both theseglandsarecloselyassociated with thenictitating membrane. The lacrimal drainagetakesplacethrough onepunctum which lieson the inner surfaceof the lowerlid, ashortdistancefrom the innercanthus. It drains into the nasolacrimalduct (Prince et al., 1961). The dogandthemonkey bothhavewell-developedlacrimal glands.The dogdoes nothaveaHarder’sglandbutdoes haveanictitan glandassociated with thenictitating membrane.Theseglandsproduceaseroussecretionwhichpasses from the lacrimal
sacinto thenasolacrimalduct.
NOW! let’stakealookat thecorneain theseanimals.The referencesin the literature which I haveconsultedgaveconflicting measurements for the thicknessof the cornea1epitheliumandfor theentire cornea,aswellasdifferencesin thesuperficial
areaof thecornea.Thesedifferencesinmeasurementcouldbeaccountedfor by the methodthat wasemployed,andwhetherthemeasurements weremadeoneyeswhich hadbeenremovedfrom theanimalsfor asufficienttimetopermit aconsiderableloss of moisture.Davcon (1962) reports: “The mostrecent measurements of the thicknessof normalcorneascarriedout in vivo by variousoptical methodsgive the followingvalues,” which I will showbelow. He alsocalculatedthesuperficialareasof
corneain relation to the total areaof theglobe (Table I). Thesemeasurements

showdifferencesin the superficialareaof the corneawhich

TABLE 1 CORNEAL THICKNESS AND CORNEAL AREAOF SEVERAL SPECIES OF AND.XAL~
Thickness Species (mm)
Man 0.53-0.54 Rabbit 0.4 Mouse 0.1 Rat -

a As percentage of the total area of the globe. Arean (k)

7 2.5
50 wl “-96 JOHN H. BECKLEY

may bemeaningful, and differences in the thickness of the cornea. To my knowledge in viva measurements by optical methods of the cornea of dogs and monkeys have not been published. The ophthalmologist, when commenting on the possible ‘Lmechanism of injurious action” to the human eye following the accidental introduction of various substances, often refers to experiments conducted on rabbit eyes. Clinically, he has observed differences in the reaction of the cornea to substances accidentally introduced into the eye which may be related to their physical character. He is aware, on the basis of published reports, that differences in pH may alter the speed and degree of penetration of the cornea1 epithelium and conjunctiva. The experimental work conducted in this regard has been done principally on normal rabbit eyes with intact cornea1 epithelium and on eyes from which the cornea1 epithelium has been removed. When the cornea1epithelium is not intact, there are considerable differences in the character of the injury to the eye. In all the reports I have seenon this type of experimentally induced eye injury on rabbits, with or without cornea1 epithelium, single compounds
were employed in various concentrations, and there was awide range in the duration of contact with the eye. We, however, are principally concerned with products which
are mixtures of substances, and not with single compounds. What have the published reports of the last two years revealed on experience with the Draize (1959) method, or modifications, as a“test of irritancy of substances to the eye mucosa”? Russell and Hoch (1962)) concerned with the reproducibility of the method, recommended two important modifications: (1) a standardization of procedure for instillation of substances into the rabbit eye, and (2) the development of a standard substance which could beused by any testing laboratory to determine whether it is scoring at a “standard” level. Olson etal. (1962), in testing several surfactants, elected to screen the rabbits for possible cornea1 injury by instilling fluorescein into the eyes 24 hours prior to use. Fluorescein was also employed as an aid in assessing cornea1 injury. The type and intensity of reactions were rated from 1 to 6, from no effect to very severe. Their system of evaluating type and intensity of reaction included not only objective observations of the eyes, but observation also of how the animals reacted. They referred to the latter as a “pain evaluation.” In this study it was interesting and maybe significant that the severity of conjunctival irritation did not necessarily correlate with cornea1 and iridial involvement. Kay and Calandra (1962) published their “Descriptive Eye Irritation Ratings,” which is so much more meaningful than the straight numerical scoring. In discussing the range of eye irritation typical of some general types of cosmetic products, the authors say: “It is worth observing that the test we have been discussing is not to be thought of as one whose results are necessarily the exact equivalent of those which might be expected following accidental contact of the test agent with the human eye. The basic test we have discussed is, comparatively speaking, a very stringent one and eye irritation following human ocular contact may be considerably reduced in intensity depending upon the particular circumstances involved. In a sense, we are attempting, via this rather rigorous rabbit test, to assess the maximum ocular irritation which might be produced in man.” Rieger and Battista (1964) state: “There is very little correlation, if any, betweenCOMPARATIVE EYE TESTING 97

the results obtained from animal tests and actual use of the product by the consumer. The Draize rabbit eye test has beenwidely used as atest of safety for products used in, on, around and above the eyes. Information has been presented indicating that different laboratories, presumably using the same techniques, have obtained conflicting results with the same product. This might indicate that the results of the rabbit test depend either on the conduct of the test, method of interpreting the results, or perhaps even on the particular group of rabbits used.” The authors suggest: “Human eye instillation tests should be conducted after rabbit eye tests to ascertain safety.” I must refer very briefly to Gaunt and Harper’s paper (1964)-a well-presented paper followed by adiscussion that was most spirited. The authors state: “The major problem in all animal screening tests

. . . is the subsequent interpretation of the recorded data in terms of the anticipated response in man. Obviously man himself must be considered the final test. Until it is established, however, that the product is safe, then asaprecautionary measure it must be assumed that the human eye will not beless sensitive than that of the animal used in the test.” During the discussion,
one of the authors, in describing the immediate reaction in one of the rabbits, said: “The eye was closed and there was slight lacrimation.” This is the first time I have
seenany reference to arabbit’s eyelacrimating. Dr. Levenstein, in his rather lengthy and most informative discussion of this paper, said: “It must be remembered that the rabbit eye is very unlike the human eye. It has tissues present which are not present in the human eye, and therefore everything that is found in the rabbit’s eye cannot be applied to the human.” Referring to the 1964 pamphlet, Principles and Procedures for Evaluating the Toxicity of Household Substances,l prepared by the Committee on Toxicology of the National Academy of Sciences, National Research Council, the recommended procedure for testing compounds to determine potential eye irritation in part says: “Albino rabbits are ideally suited for the study of compounds which may produce mild to severe eye irritation. The relatively large size of the eye, the consequent ease of instillation of test material, and the ease of the evaluation of results, commend the rabbit over other species for eye irritation tests.” The Committee goes on to suggest: “Since the rabbit eyes may frequently show evidence of injury sustained in the course of living, it is important that the eyes be examined before application of the test substance.” They suggest instillation of fluorescein, and preliminary examination to screen the animals prior to use. This type of screening procedure, as you will recall, was used by Olson et al. (1962). The Committee further recommended that, for the purposes of the Federal Hazardous Substances Labeling Act, adose of 0.1ml of liquid or 10mg of fine, dry powder be instilled into the conjunctival sac of six rabbits. The lids are to be held open for 20-30 seconds, and the animals are then returned to their cages. This procedure differs in several important respects from the current test for eye irritants prescribed in the regulations” to the Act. This brings us to our experience with a liquid product which was tested in the eyes of several animal species, including man. We use a number of well-established and, in our opinion, competent consulting laboratories for conducting animal tests on our products. We often run screening tests

1 Publication 1138, National Academy of Sciences, National Research Council, Washington, D, C, 2 21 CFR 191.12.98 JOHN H. BECKLEY

for eye irritation using three or four rabbits and have run as many as 80-100 such screening tests on variations of the same product, before using groups of six rabbits
on selected formulations. We will report on a formulation which, in the rabbit eye, produced cornea1 and iridial involvement. The same product instilled into the eye of the dog produced less overall reaction, and in the monkey still less (Table 2, Laboratory A; Table 3,Laboratory B). It is not dermally toxic or aprimary dermal irritant on rabbits. Human skin testing did not produce primary irritation or sensitivity. The test material was alight-duty liquid detergent, consisting of water, SOY,, ethyl alcohol, lZ%, and the rest an alkylbenzene sulfonate plus conventional organic foam builder and solubilizer. It contained no inorganic salts, other than a small amount of the sodium sulfate usually present inindustrial alkylbenzene sulfonates. Its pH was 6.3.

Human Findings
The human eye testing was under the direct supervision of physicians, including
an ophthalmologist. There were three separate human eye studies using this formulation by three different investigators. Fifteen male subjects were used in each study. Study A. One-tenth milliliter of a series of increasing concentrations beginning with 10% was instilled into the lower conjunctival sac of one eye. The eye was not washed out. When it was found that the diluted solutions could be tolerated, 0.1ml of the undiluted solution was instilled into the conjunctival sac daily for 7 consecutive days. There was no immediate visible reaction to any of the diluted solutions, or to the single instillation of undiluted solution. However, with the higher concentrations there
was immediate burning sensation which lasted about 20 minutes; during this time there developed a slight injection of the conjunctival vessels which abated within 1
or 2hours, and no sign of irritation was evidenced the next day. Readings were made immediately and during the first 2-3 hours, and at the end of each 24 hours. Ten of the subjects completed the study without evidence of eye damage. Five of the subjects began to develop aconjunctivitis after 3 or 4 days, and further instillations were stopped. The conjunctivitis promptly disappeared. In no instance was there cornea1 or iris involvement. Study B. This study differed from Study A in that 0.1ml of the undiluted solutions
were instilled on alternate days into the right and then the left conjunctival sac in gradually increasing concentrations until 100% concentration was achieved. In every instance the 0.1ml of fluid flooded the conjunctival sac. The solution was held in place by a gauze square for 2 minutes. Ten minutes after the removal of the gauze, the eyes were washed with water. After the twentieth day, 0.1ml of the 1005%solution was applied to the same conjunctival sac for a period of 8 consecutive days. Readings were made immediately after removal of the gauze square, and 4 and 5 hours later, and at the end of each 24-hour period. There was transient burning, discomfort, and conjunctival erythema in all subjects. These symptoms were immediate and lasted for afew minutes to afew hours. In most cases, the symptoms were more prolonged in the early stages of the study when low concentrations of the substance were used. Near the completion of the test, when higher concentrations wereCOMPARATIVE EYE TESTING 99

TABLE 2 SCORES OF OCULAR LESIONS PRODUCED BY INSTILLATION OF A LIoum DETERGENT INTO RABBIT EYES (LABORATORY A)
Time of Eyes Eyes reading Tissue unflusheda flushedh
24 hours Cornea 20.0 20.0 Iris 2.5 3.3 Conjunctiva 8.7 9.3 48 hours Cornea 16.7 13.3 Iris 3.3 3.3 Conjunctiva 6.3 9.3 72 hours Cornea 20.0 13.3 Iris 2.5 3.3 Conjunctiva 6.7 5.3 4days Cornea 20.0 13.3 Iris 25 3.3 Conjunctiva 13.0 2.0 7days Cornea 21.7 0 Iris 2.5 0 Conjunctiva 10.3 0

a The numbers in this column are the mean Draize (1959) scores of six animals. b The numbers in this column are the mean Draize (1959) scores of three animals. The eyes
were flushed 4 seconds after instillation of the sample.

TABLE 3 SCORES OF OCULAR LESIONS PRODUCED BY INSTILLATION OF .4 LIQUID DETERGENT INTO THE EYES OF SEVERAL SPECIES OF ANIMALS (LABORATORY B)
Time of reading Tissue Eyes unflusheda Eyes flushed0
Rabbit Dog Monkey Rabbit Dois Monkey
1hour

24hours

48 hours

72 hours

4days

7days Cornea

Iris Conjunctiva Cornea Iris Conjunctiva Cornea Iris Conjunctiva Cornea Iris Conjunctiva Cornea Iris Conjunctiva Cornea Iris Conjunctiva 33.3 40.0 20.0 lS.0 10.0 s.o 2.S 10.0 12.0 4.0 1.0 10.0 33.3 45.0 10.0 13..3 10.0 5.O 0 10.0 12.0 4.0 0 10.0 26.7 30.0 5.0 6.7 10.0 s.o 0 10.0 10.0 4.0 0 8.7 21.7 30.0 0 5.0 10.0 5.0 0 8.3 9.3 0 0 7.3 10.0 10.0 0 1.7 10.0 5.0 0 6.7 10.0 0 0 4.7 6.7 0 0 1.7 8.3 0 0 3.3 6.7 0 0 2.7 40.0 0 0 0 0 0 20.0 0 0 0 0 0 20.0 0 0 0 0 0 20.0 0 0 0 0 0 10.0 0 0 0 0 0 0 0 0 0 0 0

a The numbers in these columns are the mean Draize (1959) scores of six rabbits, six dogs, and four monkeys. b The numbers in these columns are the mean Draize (1959) scores of three animals of each species. The eyes were flushed 4 seconds after instillation of the sample in the case of rabbits and dogs; 2 seconds in the case of monkeys.100 JOHN H. BECKLEY

employed, reactions were minimal except in three subjects. One subject developed a conjunctival erythema which lasted one day following the instillation of the 60% concentration but tolerated the higher concentrations. Two subjects developed similar reactions, one on days 8, 9, and 16, the other on days 8, 12, and 18. In the latter there was a localized collection of purulent material on the lower conjunctiva on day 18.The erythema in this subject was still present on day 19,and the instillation
was placed in the other eye. Because the irritation seen in these two subjects might be due to superimposed bacterial infection, conjunctival cultures were obtained. NO pathogenic organisms were isolated. The ophthalmologist who took part in this study noted that the subjects who developed more pronounced reactions later in the study gave histories of low-grade pustular conjunctivitis which occasionally flared up. He felt that the symptoms and signs observed were due to fluctuations in susceptibility in these subjects. During the entire study it was observed that neither cornea nor iris
was affected. None of the subjects developed chronic conjunctivitis. There was no evidence of sensitization. Study C.A third study with 15malesubjectswasconducted.The procedurewas essentiallythe sameasthat of theprevious two studiesexcept that 3 dropsof the testmaterial wereinstilled into theeyeeachday for 3days.All thesubjectsexperiencedimmediateburning andsmarting,andhyperemiawasnoted in about 15minutes.All subjectswerenormalat the24-hourreading.Onesubjectdevelopedamore persistentconjunctivitis. Thissubjectgaveahistory of surgicalremovalofachalazion severalweeksbefore.This subjectwaseliminatedfrom thegroup. Noneof the subjectsdevelopedachronicconjunctivitis, andin nocasewasthereinvolvement of the irisor cornea. The reportswhich wehavegiven showadifferencein the resultsobtainedin the rabbit, dog,monkey, andman. Osleris reported to have said: “May I bedelivered from the doctor whodraws conclusionsbasedononecasehistory.” Wewill not besoboldorfoolish.Wecannot say why human corneaand iris werenot involved here. We could say it can be accounted for by structural differencesin the corneas,or by speciesdifference in reaction to thisparticular formulation. We don’t know. The structural differencesin the rabbit, the dog,themonkey, andthehumaneye maybesufficienttomakeit imperative that comparativestudiesbemadeusingthese species andperhapsothers.To helpexplain why thesespecies often react differently to the samesubstance,wemust have moreprecisecomparative knowledgeof the anatomy, histology, andphysiology of their eyes,particularly the cornea, iris, conjunctiva, and lacrimal andaccessoryglands. What dowe know about the “mechanismof injurious action” from substances introduced into the eyesof the rabbit, the dog, the monkey, or man?Very little. Of course,wewouldall condemndeliberately placingsubstances into thehumaneye without first knowinghowthey react toanimaleyetissue,aswewouldcondemnthe
useof any newformulation onor in thehumanbody exclusiveof the eye,without knowledgeof its reaction onorin various animalspecies. John Draize andhisassociates madeoneof thegreat contributions by &v&ping
a method,the first standardizedmethod, for establishingthe safety of products to be usedin the eyesandto estimatethe potential injury that may occur if certainCOMPARATIVE EYE TESTING 101

products are accidentally introduced into the eye. It is still the most widely used method the world over. Its shortcomings have been discussed again and again. There have been modifications. One of the modifications has become official under the Federal Hazardous Substances Labeling Act. We recommend that we quit finding fault with the prescribed method of using rabbits as the test animal and come up with a better method and amore suitable animal, if we can. This could begin by reporting
our experiences with other species of animals than the rabbit and with man. The question we should seek to answer is not only whether aproduct is potentially dangerous to the human eye,but why? And what is the “mechanism of injurious action”?

REFERENCES
DAVSON, H. (1962). The Eye. Vol. I, liegetative Physiology atid Biochemistry. Academic Press, New York. DRAIZE, J. H. (1959). Dermal toxicity. In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Assoc. Food Drug Officials U.S., .4ustin, Texas. DUKE-ELDER, S. (1961). System of Ophthalmology. Kempton, London. GAUNT, I. F., and HARPER, K. H. (1964). The potential irritancy to the rabbit eye mucosa of certain commercially available shampoos. J. Sot. Cosmetic Chemists 15, 209-230. GRANT, W. M. (1962). Toxicology of the Eye. Thomas, Springfield, Illinois. KAY, J. H., and CALANDRA, J, C. (1962). Interpretation of eye irritation tests. J. Sac. Cosmetic Chemists 13, 281-289. OLSON, K. J., DUPREE, R. W., PLOMER, E. T., and ROWE, V. K. (1962). Toxicological properties of several commercially available surfactants. J. Sot. Cosmetic Chemists 13,469-476. PRINCE, J. H., DIESEM, C. D., EGLITIS, I., and RUSKELL, G. L. (1961). Anatomy and Histology of the Eye and Orbit in Domestic Animals. Thomas, Springfield, Illinois. RIEGER, M. M., and BATTISTA, G. W. (1964). Some experiences in the safety testing of cosmetics.
J. SOL. Cosmetic Chemists l.5, 161-172. RUSSELL, K. L., and HOCH, S. G. (1962). Product development and rabbit eye irritation. proc, Sci. Sect. Toilet Goods Assoc. 37, 27-32.